ATR-FTIR Microspectroscopy Brings a Novel Insight Into the Study of Cell Wall Chemistry at the Cellular Level

Wood is a complex tissue that fulfills three major functions in trees: water conduction, mechanical support and nutrient storage. In Angiosperm trees, vessels, fibers and parenchyma rays are respectively assigned to these functions. Cell wall composition and structure strongly varies according to cell type, developmental stages and environmental conditions. This complexity can therefore hinder the study of the molecular mechanisms of wood formation, underlying the construction of its properties. However, this can be circumvented thanks to the development of cell-specific approaches and microphenotyping. Here, we present a non-destructive microphenotyping method based on attenuated total reflectance–Fourier transformed infrared (ATR-FTIR) microspectroscopy. We applied this technique to three types of poplar wood: normal wood of staked trees (NW), tension and opposite wood of artificially tilted trees (TW, OW). TW is produced by angiosperm trees in response to mechanical strains and is characterized by the presence of G fibers, exhibiting a thick gelatinous extralayer, named G-layer, located in place of the usual S2 and/or S3 layers. By contrast, OW located on the opposite side of the trunk is totally deprived of fibers with G-layers. We developed a workflow for hyperspectral image analysis with both automatic pixel clustering according to cell wall types and identification of differentially absorbed wavenumbers (DAWNs). As pixel clustering failed to assign pixels to ray S-layers with sufficient efficiency, the IR profiling and identification of DAWNs were restricted to fiber and vessel cell walls. As reported elsewhere, this workflow identified cellulose as the main component of the G-layers, while the amount in acetylated xylans and lignins were shown to be reduced. These results validate ATR-FTIR technique for in situ characterization of G layers. In addition, this study brought new information about IR profiling of S-layers in TW, OW and NW. While OW and NW exhibited similar profiles, TW fibers S-layers combined characteristics of TW G-layers and of regular fiber S-layers. Unexpectedly, vessel S-layers of the three kinds of wood showed significant differences in IR profiling. In conclusion, ATR-FTIR microspectroscopy offers new possibilities for studying cell wall composition at the cell level

ATR-FTIR imaging: phenotyping at the cell wall level in poplar wood

Trees are able to grow high and to live old thanks to the remarkable properties of their wood. As a matter of a fact, wood delivers three major functions: (1) water conduction from roots to crown, (2) support of the ever-increasing mass of the growing tree and (3) storage of temporary reserves, important for tree growth over the years. In angiosperm trees, different wood cell types are affected to each of these functions. Fibers are involved in tree mechanical support, vessels in water conduction and parenchyma rays in starch and/or lipid storage during the resting period. In addition, these cell types have distinct developmental programs. While fibers and vessels are early-dying cells, parenchyma rays stay alive longer. Therefore wood is a complex patchwork of cells and its structure results from the three-dimensional assembly of the cell walls of dead fibers and vessels, interconnected with still living parenchyma rays. This great complexity stands as an obstacle when studying wood formation and the construction of wood properties. However, this can be circumvented thanks to the development of cell-specific approaches. We developed a non-destructive method based on ATR-FTIR imaging on poplar wood sections. This technology enables to collect IR-absorbance spectra from small areas of cross-sections, which makes possible to differentiate between wood cell-types or even between the different cell wall layers from a single fiber. We first demonstrated that spectra taken from fiber cell walls on cross-sections differed from spectra obtained from wood powder. We also showed that ATR-FTIR imaging is able to discriminate the cell walls of fibers, vessels and rays. These findings are in accordance with other studies [1], but with an improved spatial resolution. ATR-FTIR microspectroscopy is thus a promising tool to finely characterize the cell wall of different wood cell types. This work has been partly supported by the OPeNSPeNU project (funded by the Centre Val de Loire Region

Conjugating immunolocalization and afm observations to determine if rhamnogalacturonan-i type pectins are responsible for the generation of maturation stress in poplar tension wood fibres

International audienceTension wood (TW) is produced by temperate hardwood trees in order to support theirincreasing weight, orient their axes and cope with environmental cues such as wind. Poplar TWfibres harbour a supplemental layer, the G-layer, rich in crystalline cellulose, containing matrixpolysaccharides but no lignin. The tensile force responsible for the specific mechanicalproperties of TW originates from the G-layer and is transmitted to cellulose microfibrils soonafter their deposition, during G-fibre maturation (Clair et al, 2011). This force is likely tooriginate from physical changes in the high porosity hydrogel recently identified in the G-layer.RG-I type pectins appear as good candidate molecules responsible for the formation of this gel.Indeed, during G-layer maturation, LM5 labelling (specific to RG-I side chains) decreased,while RU1 labelling (specific to RG-I backbone) increased (Guedes et al, 2017). This suggesteda hydrolysis of the RG-I side chains during G-fibre maturation possibly by a β-galactosidase asdemonstrated in flax phloem fibers (Roach et al, 2011). Flax phloem fibres and TW G-fibresexhibit many similar features and, in flax, it has been shown that the hydrolysis of the sidechains of RG-I type pectins was associated to the very peculiar mechanical properties of bastfibres.In order to determine if RG-I pectins were effectively involved in the building of the G-layertensile force, we carried out different measurements on a common sampling during G-fibredevelopment: i) β-galactosidase activities using a histochemical test, ii) the evolution of RG-Iimmunolabelling profiles using LM5 and RU1 as probes and iii) the stiffening of the differentcell wall layers using Atomic Force Microscopy (AFM). We found a good correlation betweenβ-galactosidase activities and RG-I immunological labelling but we failed to establish a directassociation between RG-I hydrolysis and cell wall stiffening

Conjugating immunolocalization and afm observations to determine if rhamnogalacturonan-i type pectins are responsible for the generation of maturation stress in poplar tension wood fibres

International audienceTension wood (TW) is produced by temperate hardwood trees in order to support theirincreasing weight, orient their axes and cope with environmental cues such as wind. Poplar TWfibres harbour a supplemental layer, the G-layer, rich in crystalline cellulose, containing matrixpolysaccharides but no lignin. The tensile force responsible for the specific mechanicalproperties of TW originates from the G-layer and is transmitted to cellulose microfibrils soonafter their deposition, during G-fibre maturation (Clair et al, 2011). This force is likely tooriginate from physical changes in the high porosity hydrogel recently identified in the G-layer.RG-I type pectins appear as good candidate molecules responsible for the formation of this gel.Indeed, during G-layer maturation, LM5 labelling (specific to RG-I side chains) decreased,while RU1 labelling (specific to RG-I backbone) increased (Guedes et al, 2017). This suggesteda hydrolysis of the RG-I side chains during G-fibre maturation possibly by a β-galactosidase asdemonstrated in flax phloem fibers (Roach et al, 2011). Flax phloem fibres and TW G-fibresexhibit many similar features and, in flax, it has been shown that the hydrolysis of the sidechains of RG-I type pectins was associated to the very peculiar mechanical properties of bastfibres.In order to determine if RG-I pectins were effectively involved in the building of the G-layertensile force, we carried out different measurements on a common sampling during G-fibredevelopment: i) β-galactosidase activities using a histochemical test, ii) the evolution of RG-Iimmunolabelling profiles using LM5 and RU1 as probes and iii) the stiffening of the differentcell wall layers using Atomic Force Microscopy (AFM). We found a good correlation betweenβ-galactosidase activities and RG-I immunological labelling but we failed to establish a directassociation between RG-I hydrolysis and cell wall stiffening

Non-cellulosic polysaccharide distribution during G-layer formation in poplar tension wood fibers: abundance of rhamnogalacturonan I and arabinogalactan proteins but no evidence of xyloglucan

RG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood.Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties

Both ChIP-SEQ and in planta gene modification demonstrate the involvement of MYB090 and MYB156 in secondary cell wall formation in poplar

Lakhal W., Boizot N., Lesage-Descauses M-C., Rogier O., Lainé-Prade V., Laurans F., Ader K., Charpentier J-P., Decoville M., Bénédetti H., Leplé J-C., Pilate G., Déjardin A, 2015. Both ChIP-SEQ and in planta gene modification demonstrate the involvement of MYB090 and MYB156 in secondary cell wall formation in poplar. INUPRAG Meeting, 6-8 October 2015, Nancy, France, communication orale.Lakhal W., Boizot N., Lesage-Descauses M-C., Rogier O., Lainé-Prade V., Laurans F., Ader K., Charpentier J-P., Decoville M., Bénédetti H., Leplé J-C., Pilate G., Déjardin A, 2015. Both ChIP-SEQ and in planta gene modification demonstrate the involvement of MYB090 and MYB156 in secondary cell wall formation in poplar. INUPRAG Meeting, 6-8 October 2015, Nancy, France, communication orale.Both ChIP-SEQ and in planta gene modification demonstrate the involvement of MYB090 and MYB156 in secondary cell wall formation in poplar. INUPRAG-Meeting 201

RNAseq based variant dataset in a black poplar association panel

International audienceObjective Black poplar (Populus nigra L.) is a species native to Eurasia with a wide distribution area. It is an ecologically important species from riparian ecosystems, that is used as a parent of interspecific (P. deltoides x P. nigra) cultivated poplar hybrids. Variant detection from transcriptomics sequences of 241 P. nigra individuals, sampled in natural populations from 11 river catchments (in four European countries) is described here. These data provide new valuable resources for population structure analysis, population genomics and genome-wide association studies. Data description We generated transcriptomics data from a mixture of young differentiating xylem and cambium tissues of 480 Populus nigra trees sampled in a common garden experiment located at Orléans (France), corresponding to 241 genotypes (2 clonal replicates per genotype, at maximum) by using RNAseq technology. We launched on the resulting sequences an in-silico pipeline that allowed us to obtain 878,957 biallelic polymorphisms without missing data. More than 99% of these positions are annotated and 98.8% are located on the 19 chromosomes of the P. trichocarpa reference genome. The raw RNAseq sequences are available at the NCBI Sequence Read Archive SPR188754 and the variant dataset at the Recherche Data Gouv repository under https:// doi. org/ 10. 15454/ 8DQXK5

A systems biology approach for identifying candidate genes involved in the natural variability of biomass yield and chemical properties in black poplar

Lignocellulosic biomass is a renewable resource of interest for biorefinery. However, current poplarvarieties have not been selected for this specific purpose. The factors affecting biomass yield andchemical properties need thus to be studied. With this objective, we have initiated a systems biologyapproach, integrating genomic, transcriptomic and phenotypic data in natural populations of blackpoplar (Populus nigra). Up to now, we have focused on a subset of 12 genotypes from 6 populationsand trialled in a randomized complete block design located at INRA Orléans, France. Thetranscriptome of 2 biological replicates of each genotype has been explored through RNA sequencing(RNAseq) of pools of young differentiating xylem and cambium. Additionally, biomass yield wasevaluated through measurements of height and diameter on 6 replicates of each genotype acrossseveral years and rotations, while biomass propertieswere assessed through chemical analyses oflignin, cellulose and hemicellulose concentrations as well as saccharification potential on 3 replicatesof each genotype. The resulting data were used to build a weighted gene co-expression network andidentify gene modules whose expression was correlated with biomass yield and/or quality at thegenotypic level. Remarkably, the largest module (1,460 transcripts) was significantly associated withklason lignin content and displayed an enrichment in genes involved in secondary cell wall formation.Four candidate genes from this module were further selected to validate the detected quantitative traittranscripts (QTTs) on 2 new replicates of the 12 genotypes using RT-qPCR. The resulting expressionlevels were significantly correlated to those previously quantified by RNAseq and to the klason lignincontent in the wood samples. These results demonstrate the interest of our approach, and thus opensome prospects towards the identification of new candidate genes whose functions remain to beelucidated